Abstract

Distinct types of GABAergic interneurons target different subcellular domains of pyramidal cells, thereby shaping pyramidal cell activity patterns. Whether the presynaptic heterogeneity of GABAergic innervation is mirrored by specific postsynaptic factors is largely unexplored. Here we show that dystroglycan, a protein responsible for the majority of congenital muscular dystrophies when dysfunctional, has a function at postsynaptic sites restricted to a subset of GABAergic interneurons. Conditional deletion of Dag1, encoding dystroglycan, in pyramidal cells caused loss of CCK-positive basket cell terminals in hippocampus and neocortex. PV-positive basket cell terminals were unaffected in mutant mice, demonstrating interneuron subtype-specific function of dystroglycan. Loss of dystroglycan in pyramidal cells had little influence on clustering of other GABAergic postsynaptic proteins and of glutamatergic synaptic proteins. CCK-positive terminals were not established at P21 in the absence of dystroglycan and were markedly reduced when dystroglycan was ablated in adult mice, suggesting a role for dystroglycan in both formation and maintenance of CCK-positive terminals. The necessity of neuronal dystroglycan for functional innervation by CCK-positive basket cell axon terminals was confirmed by reduced frequency of inhibitory events in pyramidal cells of dystroglycan-deficient mice and further corroborated by the inefficiency of carbachol to increase IPSC frequency in these cells. Finally, neurexin binding seems dispensable for dystroglycan function because knock-in mice expressing binding-deficient T190M dystroglycan displayed normal CCK-positive terminals. Together, we describe a novel function of dystroglycan in interneuron subtype-specific trans-synaptic signaling, revealing correlation of presynaptic and postsynaptic molecular diversity.

SIGNIFICANCE STATEMENT:

Dystroglycan, an extracellular and transmembrane protein of the dystrophin-glycoprotein complex, is at the center of molecular studies of muscular dystrophies. Although its synaptic distribution in cortical brain regions is long established, function of dystroglycan in the synapse remained obscure. Using mice that selectively lack neuronal dystroglycan, we provide evidence that a subset of GABAergic interneurons requires dystroglycan for formation and maintenance of axonal terminals on pyramidal cells. As such, dystroglycan is the first postsynaptic GABAergic protein for which an interneuron terminal-specific function could be shown. Our findings also offer a new perspective on the mechanisms that lead to intellectual disability in muscular dystrophies without associated brain malformations.

Characterization of NEX-Cre/Dag1 conditional KO mice. A, Representative examples of NEX-CreTg/+/Dag1loxP/+ (control) and NEX-CreTg/+/Dag1loxP/loxP (cKO) mice (4 months of age, siblings, both female). B, cKO mice exhibit reduced body weight compared with sibling control mice. C, Wet brain weight was lower in cKO mice than in controls. D, cKO mice exhibited a higher mortality rate than control mice, resulting in a frequency of cKO mice lower than the expected 25% at the age of 10 weeks. E, Similar levels of α-DG isolated from cheek muscle were found for cKO and control mice. F, Cre expression was restricted to pyramidal cells in the hippocampus of cKO and control mice. In adult mice, dentate gyrus granule cells were not immunoreactive for Cre recombinase. NeuN and DAPI labeling shows intact neuronal migration when NEX-Cre is used as driver line to ablate Dag1. G, In primary somatosensory cortex, Cre expression was also restricted to pyramidal cells. No migratory deficits were found in the neocortex in cKO mice. ***p < 0.001.

Loss of neuronal dystroglycan does not prohibit formation of GABAergic PSD but leads to minor changes in GABAAR subunit clustering. A–C, Triple immunofluorescence labeling of GABAergic postsynaptic markers in pyramidal layer of hippocampus CA1 area. The DGC is largely colocalized with α2 subunit and VGAT (A; arrowheads) but also with α1 subunit and NL2 (B; arrowheads). A minority of DGC clusters is not associated with GABAergic markers (A, B; arrows). D–H, Quantification of postsynaptic GABAergic markers in CA1 pyramidal cell layer. Cluster density and size are shown for GABAAR α1 (D), α2 (F), and γ2 (H) subunits and for gephyrin (E) and NL2 (G). A decrease of α1 subunit cluster size was accompanied by an increased α2 subunit cluster density. I, Colocalization of postsynaptic GABAergic markers was analyzed in cKO and control mice. Data are the number of colocalized clusters as percentage of first mentioned marker. No significant differences in colocalization were found between genotypes. J, Clustering of synArfGEF was analyzed in CA1 pyramidal layer of DG cKO and control mice. Data points represent individual mice (for statistical tests, see ). **p < 0.01. ***p < 0.001.

Neuronal dystroglycan ablation leads to specific loss of terminals from CCK-positive basket cells on pyramidal cells in neocortex. A–C, Triple immunofluorescence labeling of GABAergic markers in layer 2/3 of primary somatosensory cortex (S1) of DG cKO and control mice. A, As in hippocampus, the majority of DG clusters is colocalized with presynaptic and postsynaptic GABAergic markers in neocortex. B, Neocortical PV and VGluT3 immunolabeling is not affected by loss of neuronal DG. C, CCK8 and CB1 immunofluorescence is strongly reduced in neocortex of DG cKO mice. Immunolabeling of synArfGEF showed clustered distribution and did not differ between genotypes. D, Overview of S1 of DG cKO and control mice. Typical punctate CB1 immunofluorescence was lost across all layers of the cortex in DG cKO mice. E–I, Quantification of presynaptic GABAergic markers in S1 layer 2/3. VGAT and PV, and in contrast to hippocampus, also VGluT3 were not reduced in density in mice lacking neuronal DG (E–G). However, CB1 and CCK8 showed a similar reduction as in hippocampus in DG cKO mice compared with control mice (H, I). Data points represent individual mice (for statistical tests, see ). **p < 0.01. ***p < 0.001.

Neuronal dystroglycan is not necessary for clustering of glutamatergic synaptic proteins. A, B, To assess integrity of glutamatergic postsynaptic structures, antibodies to PSD-95 and bassoon were used and immunofluorescence quantified in stratum pyramidale and stratum radiatum. Cluster density and size were analyzed in stratum pyramidale and fluorescence intensity in stratum radiatum. All parameters analyzed did not differ substantially between genotypes. C, VGluT1 was used as a marker of glutamatergic presynaptic terminals, and puncta density and size in stratum pyramidale were quantified. No changes in VGluT1 puncta density and size were found between genotypes. D, PSD-95 apposition to VGluT1 was examined in stratum pyramidale and represented as percent PSD-95 clusters apposed to VGluT1 puncta. The apposition of PSD-95 to VGluT1 did not differ between genotypes. Data points represent individual mice (for statistical tests, see ). *p < 0.05. ***p < 0.001.

Frequency and amplitude of sIPSCs are reduced in dystroglycan cKO pyramidal cells. A, Position of the recording pipette in the hippocampal CA1 region (left, 4×), and an example of a typical CA1 pyramidal cell identified using LED illumination (AlexaFluor-488, right, 40×). B, Representative example traces of whole-cell sIPSC recordings from control mice (left) and DG cKO mice (right). Average sIPSCs are shown above the traces. C, Cumulative frequency plot of IEIs of sIPSCs from control (blue line) and DG cKO cell (red line) from the traces in B (left) and cumulative frequency plot of sIPSC amplitudes from the same cells (right). D, Comparison of average sIPSC frequency and amplitude between control and DG cKO slices. DG cKO mice exhibit significantly lower sIPSC frequency and amplitude than control mice. Data points represent individual cells. *p < 0.05.

Dystroglycan is necessary for CCh-induced increase of inhibitory currents in pyramidal cells. A, Representative example traces of sIPSC recordings before (baseline, left trace) and after the application of CCh (right) in control mice. Average sIPSCs are shown above the traces. B, Cumulative frequency plots of IEIs and amplitudes of sIPSCs from traces in A. C, Comparison of average sIPSC frequency and amplitude before and after application of CCh in control slices. Application of CCh resulted in typical increase of IPSC frequency in control pyramidal cells, but amplitude was not affected by CCh. D, Representative example traces of sIPSC recordings before (baseline, left trace) and after the application of CCh (right trace) in DG cKO mice. Average sIPSCs are shown above the traces. E, Cumulative frequency plots of IEIs and amplitudes of sIPSCs from traces in D. F, Comparison of average sIPSC frequency and amplitude before and after application of CCh in DG cKO slices. In contrast to control slices, application of CCh did not lead to a significant increase of sIPSC frequency in DG cKO pyramidal cells. Data points represent individual cells. **p < 0.01.